Introduction
Patients with hematological disorders are at a higher risk of invasive fungal disease (IFD) due to various factors such as chemotherapy or transplantation, persistent neutropenia, immunosuppression, molecular targeted therapy, deep venous catheter placement, and use of broad-spectrum antibiotics [ 1 ]. Studies have shown that 10% of patients with hematological malignancies develop life-threatening IFDs during treatment, with a mortality rate approaching 29% [ 2 ].
According to the European Organization for Research and Treatment of Cancer/Mycoses Study Group Education & Research Consortium (EORTC/MSGERC) guideline [ 3 ], the opportunistic diagnosis of IFD is categorized into three levels as follows: proven IFD, probable IFD, and possible IFD. Sterile body fluid culture and histopathological identification remain the “gold standard” for the diagnosis of proven IFD; however, their feasibility and positivity rates remain unsatisfactory, and existing diagnostic methods are limited [ 4 ]. Therefore, early and precise identification of fungal pathogens carries great importance for the selection of treatment regimens and in improving outcomes in patients with hematological disorders combined with IFD.
Metagenomic next-generation sequencing (mNGS) is a new form of testing widely used for detection of viruses, bacteria, and fungi in a variety of host samples and can assist in diagnosing pathogens rapidly, thus enabling precision treatment of infectious diseases [ 5 ], which has been recommended as a useful addition for mycological evidence by the EORTC-MSG guideline [ 3 ]. In order to gain an in-depth understanding of the clinical characteristics and related risk factors of patients with hematological disorders diagnosed with IFD, the present study was conducted as a retrospective analysis of the case data of patients with hematological malignancies diagnosed with IFD.
Data and methods
Data
Patients admitted to our Hematology Department between January 1, 2023, and January 1, 2025, were selected. The study was approved by the Institutional Review Board of the hospital (Ethics Approval Number: 2023-LY-kt097). In total, 67 patients were enrolled, of which 32 comprised the IFD group and 35 comprised the non-IFD group.
Inclusion and exclusion criteria
The inclusion criteria were as follows: (i) patients diagnosed with hematological disorders in accordance with the criteria of the hematological disease diagnostic and treatment specifications, and (ii) diagnosis of IFD in accordance with the EORTC/MSGERC criteria, the Infectious Diseases Society of America (IDSA) guidelines, and the guidelines of the European Conference on Infections in Leukemia (ECIL) [ 3 ].
The exclusion criteria were inconsistent diagnosis or incomplete clinical data.
Observational indicators
The complete blood count, biochemical indicators, C-reactive protein, cytokine testing results, lymphocyte subset testing results, G test, GM test, mNGS results, and fungal and microbial culture results of all included patients were observed.
Statistics
SPSS 23.0 and R Studio software were used for statistical analysis. The independent-sample t -test was used for measurement data conforming to the normal distribution, non-parametric tests were used for measurement data not conforming to the normal distribution, and the χ 2 test was used for count data. To exclude the effect of confounding factors, risk factors with P < 0.05 in the univariate analysis were entered into multivariate logistic regression analysis.
Results
General characteristics
The IFD group consisted of 32 patients with a median age of 41 years (range: 5-84 years), of which 21 were male and 11 were female. The clinical diagnosis was aplastic anemia in five cases, myelodysplastic syndromes in five cases, leukemia in 10 cases, lymphoma in two cases, myeloma in two cases, myelofibrosis in four cases, and other diseases in four cases.
The non-IFD group consisted of 35 patients with a median age of 62 years (range: 4-88 years), of whom 20 were male and 15 were female. The clinical diagnosis was aplastic anemia in nine cases, myelodysplastic syndromes in eight cases, leukemia in 11 cases, lymphoma in two cases, multiple myeloma in one case, myelofibrosis in one case, and other diseases in three cases. The difference between the two groups was not statistically significant ( P >0.05) ( Table 1 ).
Table 1
General characteristics of patients in the IFD group and non-IFD group.
IFD, invasive fungal disease.
| Characteristic | Experimental group (n = 32) | Control group (n = 35) | P | |
| Sex | Male | 21 | 20 | 0.47 |
| Female | 11 | 15 | ||
| Age (median/range) | 41 (5-84) | 62 (4-88) | 0.45 | |
| Clinical diagnosis | Aplastic anemia | 5 | 9 | 0.67 |
| MDS | 5 | 8 | ||
| Leukemia | 10 | 11 | ||
| Lymphoma | 2 | 2 | ||
| Multiple Myeloma | 2 | 1 | ||
| Myelofibrosis | 4 | 1 | ||
| Other | 4 | 3 |
Fungal etiological diagnosis and detection
Among 67 patients, 32 were detected with fungal pathogens by mNGS, with a fungal detection rate of 47.76% (32/67). Ten patients were detected with fungal pathogens by microbiological culture, with a fungal detection rate of 14.93% (10/67), showing a statistically significant difference compared to mNGS ( P = 3.4×10 –5 , P <0.05). Eight patients tested positive in the G test, with a detection rate of 11.94% (8/67), showing a statistically significant difference compared to mNGS ( P = 5×10 –6 , P <0.05). Two patients tested positive in the GM test, with a fungal detection rate of 2.99% (2/67), showing a statistically significant difference compared to mNGS (p = 1.99×10 –9 , P <0.05).
Sources of mNGS specimens and distribution of fungal strains in the IFD group
In the IFD group, bronchoalveolar lavage fluid was used for mNGS in 16 patients, blood in 12 patients, and pleural fluid and ascites in four patients. In the non-IFD group, bronchoalveolar lavage fluid was used for mNGS in 11 patients, blood in 25 patients, and pleural fluid and ascites in two patients. The differences between the two groups were not statistically significant ( P = 0.058 > 0.05).
In the IFD group, Candida was detected in 15 cases (46.88%), Aspergillus in six cases (18.75%), Pneumocystis in four cases (12.5%), Rhizomucor in four cases (12.5%), Saccharomyces in two cases (6.25%), and Malassezia in one case (3.12%) ( Figure 1 ).
Analysis of factors associated with infection in IFD patients
The sex, age, history of diabetes mellitus, degree of neutropenia at the time of initial diagnosis, duration of neutropenia, lymphocyte count, C-reactive protein, cytokines, cluster of differentiation (CD4 + ) T cell count, and presence of concomitant bacterial infection in the IFD and non-IFD groups were included in univariate analysis using SPSS 23.0 software. The results suggested that the differences in the duration of neutropenia, C-reactive protein, CD4+ T cell count, interleukin (IL)-6, IL-10, and albumin level were statistically significant ( P <0.05) ( Table 2 ).
Table 2
Univariate analysis of factors associated with invasive fungal disease.
IFN, interferon; IL, interleukin.
| Clinical characteristic | Experimental group | Control group | P | |
| Age | >60 years | 19 | 19 | 0.675 |
| ≤60 years | 13 | 16 | ||
| Diabetes | Yes | 9 | 11 | 0.769 |
| No | 23 | 24 | ||
| Neutropenia at initial diagnosis | Yes | 8 | 8 | 0.837 |
| No | 24 | 27 | ||
| Duration of neutropenia | >10 days | 13 | 5 | 0.015 |
| ≤10 days | 19 | 30 | ||
| Elevated C-reactive protein | Yes | 24 | 17 | 0.027 |
| No | 8 | 18 | ||
| Lymphocytopenia | Yes | 13 | 11 | 0.433 |
| No | 19 | 24 | ||
| Thrombocytopenia | Yes | 21 | 27 | 0.296 |
| No | 11 | 8 | ||
| Elevated lactate dehydrogenase | Yes | 17 | 15 | 0.401 |
| No | 15 | 20 | ||
| Clusters of differentiations 4+ T cell count | ≥400/µl | 7 | 24 | 8.9×10 –5 |
| <400/µl | 26 | 11 | ||
| Elevated IL-2 | Yes | 0 | 1 | 0.335 |
| No | 32 | 34 | ||
| Elevated IL-4 | Yes | 0 | 1 | 0.335 |
| No | 32 | 34 | ||
| Elevated IL-6 | Yes | 24 | 12 | 0.001 |
| No | 8 | 23 | ||
| Elevated IL-8 | Yes | 15 | 10 | 0.122 |
| No | 17 | 25 | ||
| Elevated IL-10 | Yes | 13 | 6 | 0.033 |
| No | 19 | 29 | ||
| Elevated tumor necrosis factor-α | Yes | 3 | 3 | 0.908 |
| No | 29 | 32 | ||
| Elevated IFN-α | Yes | 1 | 2 | 0.609 |
| No | 31 | 33 | ||
| Elevated IFN-γ | Yes | 2 | 0 | 0.133 |
| No | 30 | 35 | ||
| Albumin | >30 g/l | 23 | 33 | 0.013 |
| ≤30 g/l | 9 | 2 |
Multivariate logistic regression analysis of the six risk factors with P <0.05 in the univariate analysis was performed using R 4.2.1 software. The results indicated that CD4+ T cell count <400 cells/µL (odds ratio [OR] = 9.45, P = 8.9×10 –5 ), elevated C-reactive protein (OR = 3.18, P = 0.027), elevated IL-6 (OR = 5.75, P = 0.001), elevated IL-10 (OR = 3.31, P = 0.033), hypoproteinemia (OR = 42.17, P = 0.013), and neutropenia persisting for >10 days (OR = 4.11, P = 0.015) were independent risk factors for IFD infection in patients with hematological disorders ( Figure 2 ).
These independent risk factors for IFD were used to construct a nomogram using R 4.2.1 software. The nomogram had a C-index of 0.862 with a 95% confidence interval of 0.772-0.951, indicating that it could reflect the risk factors of IFD infection in patients with hematological disorders and predict the chance of IFD infection in these patients ( Figure 3 ).
Discussion
IFD is one of the major complications in the diagnosis and treatment of malignant hematological disorders [ 6 ]. The overall incidence of IFD is 2.1% in patients with hematological malignancies undergoing chemotherapy and 26.7% in patients undergoing hematopoietic stem cell transplantation [ 7 ]. IFD remains a major cause of death in hematological disorders [ 8 ]. Studies have shown that the overall mortality rate for patients with hematological disorders treated with chemotherapy is only 1.5%, whereas the mortality rate for patients with IFD is as high as 11.7% [ 7 ]. Therefore, IFD severely impacts the long-term survival of patients with hematological disorders. The development of timely, effective, and standardized prevention and treatment strategies for IFD is crucial for clinical purposes.
Due to the atypical clinical features of IFD, sterile body fluid culture and histopathological identification remain the “gold standard” for diagnosis. However, fungal culture is time-consuming and has a low positive rate; therefore, the diagnosis of IFD is still based on stratified criteria [ 4 ]. In contrast, mNGS can detect a wide range of pathogenic microorganisms in an unbiased manner, thus providing reference for the precise clinical diagnosis and treatment of acute and critical illnesses and complex infectious diseases [ 5 ]. In the present study, the positive detection rates of fungal microbiological culture and mNGS were compared; the results showed that mNGS exhibited a higher detection rate for fungal pathogens compared to conventional culture, suggesting that mNGS has a strong advantage in the diagnosis of fungal pathogens.
Before the introduction of routine systemic prophylactic antifungal therapy in the clinic, Candida was the most common pathogen in IFD [ 9 ]. With the widespread use of fluconazole prophylaxis, the incidence of Aspergillus infection has gradually increased [ 9 ]. In recent years, with the application of new broad-spectrum triazoles, the incidence of infection by rare fungi, such as Mucor and Rhizomucor, has also increased [ 9 ]. In the present study, mNGS testing of patients with hematological disorders revealed that Candida (15 cases, 46.88%) and Aspergillus (six cases, 18.75 %) infections were the most prevalent, followed by Rhizomucor (four cases, 12.5%) and Sporothrix (four cases, 12.5%), which is generally consistent with previous findings.
Antifungal prophylaxis is a protective factor for IFD and reduces the risk of IFD and death to some extent [ 2 ]. Studies have suggested a benefit from antifungal prophylaxis in populations with ≥5% prevalence of IFD and a significant benefit in those at ≥10% high risk for IFD [ 10 ]. Therefore, patients with risk factors for IFD should receive antifungal prophylaxis, the course of which depends on whether the patient’s risk factors for IFD improve [ 8 ].
In the present study, CD4+T cell count < 400 cells/µL (OR = 7.43, P = 8.9×10 –5 ), elevated C-reactive protein (OR = 3.18, P = 0.027), elevated IL-6 (OR = 5.75, P = 0.001), elevated IL-10 (OR = 3.31, P = 0.033), hypoproteinemia (OR = 42.17, P = 0.013), and neutropenia lasting for more than 10 days (OR = 4.11, P = 0.015) were found to be independent risk factors for IFD. First, neutropenia persisting for >10 days and hypoproteinemia have been previously shown to be independent risk factors for IFD [ 7 , 11 ]; neutropenia persisting for >10 days can weaken the immune system and increase the risk of opportunistic fungal infections [ 7 , 11 ], and hypoproteinemia can lead to weakening of the immune system and impairment of the mucosal barrier of the body, which increases the risk of IFD infection [ 7 , 11 ]. Second, CD4+ T cells can play a direct antifungal role by releasing cytokines and mediating B-cell immunity [ 12 ]; therefore, their role in host defense against fungi cannot be neglected. Thus, decreased CD4+ T cells may exacerbate fungal infections in patients. Furthermore, C-reactive protein is one of the most common markers of inflammation as it can be elevated during injury, infection, and inflammation. Sidharta et al. [ 13 ] retrospectively analyzed the correlation between C-reactive protein and fungal infections in 61 patients with acute leukemia, and the results suggest that elevated C-reactive protein can be used as a marker for the screening of fungal infections. In addition, IL-6 is a cytokine, or intercellular signaling molecule, that plays an important role in the inflammatory immune response and other immune processes, including regulation of the immune response, inflammatory response, thermoregulation, and cell proliferation. In a study of 106 patients with hematological disorders, Rawlings et al. [ 14 ] found elevated levels of IL-6 in the blood and bronchoalveolar lavage fluid of patients with IFD, particularly cases involving Aspergillus. Thus, IL-6 can be used as a clinical predictor for the long-term prognosis and mortality of patients with hematological disorders. In addition, IL-10 has major multifunctional roles in modulating the immune response to fungal infections. Antachopoulos et al. [ 15 ] identified a key role for IL-10 in modulating normal host resistance to fungal pathogens using a mouse model of Aspergillus infection.
This study has some potential limitations. First, due to ethical considerations, a randomized control population could not be established. Second, based on time and resource limitations, the sample size of the present study is relatively small, and the observational indicators are insufficiently comprehensive; while sustained neutropenia is a documented host risk factor for invasive aspergillosis, and low CD4 T cell count is a risk factor for pneumocystis, the other elevated markers (e.g., elevated IL-6) are non-specific for IFD. Thus, it is necessary to adjust the study design and increase the sample size in future studies to further investigate the high-risk factors for IFD infection in patients with hematological disorders and provide a more reliable clinical foundation.
In conclusion, it is important to understand the clinical characteristics of IFD combined with hematological diseases, to focus on early intervention in groups at high risk of invasive mycoses, to fully leverage modern and advanced microbiological testing, to improve the early diagnosis rate of IFD, and to formulate rational antifungal treatment protocols to improve patient outcomes and quality of life.
Declaration of competing interest
The authors have no competing interests to declare.
Acknowledgments
Funding
This study was supported by the Municipal Health Commission of Nantong (No. MS2024100).
Author contributions
Xi Chen and Hongchao Qin contributed equally to this work. They wrote the manuscript, Yun Lian and Yuhua Song performed the data collection and analysis. Qiqiang Long made contributions to the study design. All the authors read and approved the final manuscript.
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